The faithful duplication of the genetic material is fundamental to all biological systems. For viruses as for cells, a complex repertoire of proteins mediates the carefully regulated process of DNA synthesis and coordinates it to the related processes of recombination and repair. The efficiency of replication is enhanced by the establishment of dedicated replication foci, and replication is regulated by networks of protein-protein interactions. In this proposal, we present our plan for continued investigation of how vaccinia virus, the prototypic poxvirus, coordinates the replication of its genome. Poxviruses are the only DNA viruses that replicate in the cytoplasm of infected cells. This autonomy from the host nucleus poses unusual challenges: the virus must encode a complete replication apparatus and must establish cytoplasmic sites for replication. Poxviruses such as vaccinia virus, variola virus, and monkeypox represent intriguing model systems as well as being of significant biomedical importance. The significance of these studies is enhanced by the importance of the viral replication proteins as targets for rational antiviral therapies. During the next funding period, we will pursue two aims, utilizing genetic, biochemical and cell biological approaches to further elucidate the replication of vaccinia virus. AIM I: Genetic and biochemical analysis of the core replication machinery. Genetic and biochemical analyses have identified a repertoire of proteins involved in vaccinia DNA replication. Our goal for these studies is to understand the structure and function of the core replication machinery. The polymerase holoenzyme, comprised of the E9 DNA polymerase/A20 processivity factor/ D4 uracil DNA glycosylase will be the major topic of study. Our investigations will focus on the mechanism of polymerase processivity and the coupling of replication and repair. The roles played by the essential I3 and H5 replication proteins will also be addressed. AIM II: Analysis of the D5 primase/helicase and reevaluation of the mechanism of DNA replication. We will continue our structure/function analysis of the essential D5 ATPase, which is predicted to be a superfamily III helicase and has recently been shown to have primase activity. These insights into the activities associated with D5 have prompted us to revisit the mechanism of poxviral replication and assess the possible involvement of both leading and lagging strand synthesis.